Multiprotein complexes mediate the bulk of biological processes. A crucial part of our understanding of these processes is therefore based on knowing which proteins interact with a protein of interest. This knowledge is extensive for oligomeric proteins whose subunit interactions are strong enough to withstand in vitro conditions. However, many oligomeric complexes, while relevant physiologically, are either transient, intrinsically unstable, or are destabilized upon dilution, depletion of cofactors and other perturbations that accompany a transition from in vivo to in vitro conditions. In part as a result of this difficulty, the existing knowledge encompasses but a small fraction of the actually occurring, physiologically relevant protein-protein interactions even among the best understood organisms. Moreover, even for proteins whose in vivo protein ligands are partly known, this knowledge is often of a qualitative kind; it rarely includes the actual affinities, let alone kinetic aspects of an in vivo interaction. Limitations of the existing in vivo methods are a major reason for this impasse.
Assays for in vivo protein interactions include crosslinking of interacting proteins with a cell-penetrating agent such as formaldehyde, and use of fluorescence resonance energy transfer to follow the interactions of dye-coupled proteins microinjected into living cells. Genetic analyses of in vivo protein interactions include searches for extragenic suppressor mutations or synthetic lethal mutations, which occur in genes whose products are at least functionally (and often physically) associated with a gene product of interest. A more recent approach, the two-hybrid technique, is based on expressing one test protein as a fusion to a DNA-binding domain of a transcriptional activator, and expressing another test protein as a fusion to a transcriptional activation domain. If the test proteins interact in vivo, a transcriptional activator is reconstituted, resulting in the induction of a reporter gene.
The repertoire of existing assays for in vivo protein interactions, while expanding, is deficient in that significant questions cannot be answered due to technical limitations. New techniques which fill these experimental voids would represent important steps forward in the art.